Early Childhood Education and Care (ECEC) settings have a pivotal role in shaping children’s dietary food habits by providing the contextual environment within which they develop these behaviours. This study examines systematic reviews for (1) the effectiveness of interventions to promote healthy eating in children aged 2–5 years attending centre-based childcare; (2) intervention characteristics which are associated with promoting healthy eating and; (3) recommendations for child-health policies and practices. An Umbrella review of systematic reviews was undertaken using a standardized search strategy in ten databases. Twelve systematic reviews were examined using validated critical appraisal and data extraction tools. Children’s dietary food intake and food choices were significantly influenced. Interventions to prevent obesity did not significantly change children’s anthropometric measures or had mixed results. Evidence was more convincing if interventions were multi-component, addressed physical activity and diet, targeted individual-level and environmental-level determinants and engaged parents. Positive outcomes were mostly facilitated by researchers/external experts and these results were not replicated when implemented in centres by ECEC providers without this support. The translation of expert-led interventions into practice warrants further exploration of implementation drivers and barriers. Based on the evidence reviewed, recommendations are made to inform child-health directed practices and policies. Good nutrition in early childhood is essential to ensure children reach their growth and developmental potential . FurtherConsiderable public health effort and research have been directed towards nutrition interventions in the home and the school setting ,14. HoweAlthough the home is still the primary influence ,22, centUmbrella reviews are becoming relatively common ,32 as a The primary aims of this Umbrella review are to examine previously published systematic reviews to determine (1) the effectiveness of interventions to promote healthy eating in children aged 2–5 years attending centre-based childcare; (2) intervention characteristics which are associated with successfully promoting healthy eating in pre-schoolers; and (3) recommendations for child-health directed policies and practices.To identify possible systematic reviews the online bibliographic databases Medline, Emcare , PsycINFO , Embase , CINAHL , Health Technology Assessment Database, ERIC, Scopus, Web of Science Core Collection, Joanna Briggs Institute (JBI) Evidence-Based Practice Database of Systematic Reviews and Cochrane Database of Systematic Reviews were searched for reviews published between January 2000 and September 2017. The search strategy is available as The following reviews were excluded: (1) studies with infants or studies where the children were attending compulsory schooling usually six years or older; (2) studies treating children for obesity or a clinical related condition; (3) studies using school, the home or settings which are not registered childcare; (4) studies in which dietary behaviour or dietary-related outcomes were secondary outcomes and not separately reported; (5) studies focused on low-income countries. Although the search strategy did not limit studies to particular countries, only systematic reviews relating to high-income countries as defined by the OECD (2017) were included because the childcare arrangements and practices are similar. An a priori protocol for the Umbrella review was registered with PROSPERO (CRD42017078749). To assess the methodological quality of the reviews and to determine the extent to which reviews had addressed the possibility of bias in the design, conduct and analysis, the Johanna Briggs Institute (JBI) Critical Appraisal Checklist for Systematic Reviews and Research Syntheses was used . This vaTo guide the extraction and synthesis of data from the selected studies and minimize the risk of author bias, a standardized tool, the JBI Data Extraction Form for Systematic Reviews and Research Synthesis  was emplp < 0.0005).The study selection process is summarised in Twelve systematic reviews were included in the final review and the quality assessment ratings are tabulated in Most of the primary studies in all of the reviews were randomised control trials (RCT) or cluster-RCT followed by case-control trials or quasi-experimental studies . ConcernThere was considerable heterogeneity between primary studies which precluded pooling of the data and meta-analysis or any systematic reviews undertaking Grading of Recommendations Assessment, Development and Evaluation (GRADE) . HeterogStudy findings favoured dietary effectiveness in most of the included reviews . AssesseSeven reviews focused on obesity-prevention and obesogenic behaviours (including diet-related behaviours). Despite reporting significant effects on BMI and other measures of adiposity for some primary studies, review authors concluded overall that diet-related interventions did not have a consistently positive impact . Two other reviews reported no significant changes in weight status . Ling, Robbins et al. (2016) and Zhou, Emerson et al. (2014) reported that the primary studies which significantly affected weight outcomes were multi-component interventions which addressed both dietary and physical activity behaviours. Mikkelsen, Husby et al. (2014) reported that single component interventions did not have a significant effect on children’s fruit and vegetable intake but five of six multi-component interventions did. Actively involved and engaged parents were also associated with consistently positive impacts on children’s weight status .More positive outcomes were seen in reviews assessing interventions directed at both environmental- and individual-level determinants of healthy eating behaviours. Most effective were multi-level interventions targeting environmental-level determinants including implementation support ,55,58,61Three reviews reported that the strongest effects came from interventions targeting environmental-level determinants. Bell and Golley (2015) examined 13 primary studies, with 12 reporting significant improvement in the food provided in centres (through food policy and changes in educators’ practices), the nutritional quality of menus and parental food provision of lunchboxes. Primary studies on interventions focusing on environmental-level factors reported positive outcomes including food and nutrition policies and the food environment, however few of these studies also reported on whether children’s dietary intake had changed as a result . Wolfendn = 15). Six primary studies attributed high parental engagement to the successful achievement of their primary outcome to effect changes in children’s weight .Half of the reviews reported an association between parental involvement and engagement, and achievement of objectives in ECEC interventions ,54,55,58Positive outcomes for healthy eating behaviours were mostly reported for interventions delivered by researchers or external experts ,52,55,56Eight reviews reported the number of included primary studies which used a theoretical framework ,57,58,61Most of the dietary-related interventions targeted educators’ behaviours and practices and included nutrition education and training sessions . EducatiEffective interventions involving children included interactive educational activities as part of the childcare curriculum ,54,57,58Active parental involvement included participation in any intervention component such as receiving written material, receiving regular newsletters, attending education sessions or workshops, completing homework tasks, participating in curriculum planning or participating in interactive hands-on activities such as cooking, growing vegetables or similar activities, with their children ,54,58,60Recommendations for practice and policy  includedSummarised recommendations for future research  includedThis Umbrella review investigated the effectiveness of interventions to promote healthy eating in children aged 2–5 years attending centre-based childcare. The aim was to also identify characteristics of successful interventions and list and summarise the most frequent recommendations for policy, practice and research. Overall, 12 systematic reviews of acceptable methodological quality were included examining 101 primary studies.Despite the considerable heterogeneity, the review findings supported the proposition that interventions to promote healthy eating in children aged 2–5 years attending centre-based childcare are effective. Successful interventions were multi-component, multi-level targeting both environmental and individual-level determinants of healthy eating behaviours. Multi-component interventions included educational strategies, changes to the centre-environment and policy. These findings are consistent with the conclusions of other Umbrella reviews for other settings ,66,67,68A common recommendation in the reviews was to underpin intervention design with theoretical frameworks and effective behavioural change theories, ideally components of Social Cognitive Theory  alongsidThe translation of changes in educators’ knowledge, practices and centre environment to children’s dietary behaviours was however not consistently observed ,54. MoreMore studies assessing the dietary-related outcomes from involving and engaging with parents are required. Even small levels of parental involvement were associated with better weight status outcomes ,53,54,55More research is needed to understand the interactions between educators and parents and the impact of collaborative parental engagement. With many children spending time in childcare and the premise that all food preferences are learnt, educators’ roles are crucial as very young children are dependent upon them not only to provide food but also to guide and shape their food preferences and dietary habits . QualitaThe impact of nutrition-related strategies to build the capacity of children is also an evidence gap. Findings emphasized the importance of targeting children with interactive education and hands-on experiences which are age-appropriate ,52. ThisThe impact of nutrition-related interventions and practices on children from low socio-economic areas is of particular interest. Many of the primary studies were directed at centres in low socio-economic areas or centres with a high proportion of children from disadvantaged families. The outcomes suggest that interventions supporting these populations could help reduce health inequalities . This obMissing from this Umbrella review was evidence of the sustainability of dietary-related interventions as few primary studies were implemented for more than a year and/or outcomes measured after the intervention. The recommended duration is at least one year, ideally 1–2 years ,58. NotaAlthough the more comprehensive the intervention, the more likely it is to be successful, comprehensiveness may affect feasibility and fidelity negatively and warrants further exploration. Ward, Welker et al. (2016) found an inverse relationship between comprehensiveness and positive outcomes. Furthermore, in an Umbrella review investigating community-based interventions promoting healthy eating and physical activity, multi-component interventions were not correlated with positive outcomes . MoreoveLastly, reviewers recommended that cost-effective studies be undertaken ,56,61. LBased on the data reported in the 12 reviews, reviewers cautioned that many primary studies were rated as weak or having insufficient information to permit evaluation. The actual effect of the intervention may therefore be smaller than the effects reported because of the low quality of reporting  and geneSome of the challenges identified by Pollock et al. (2017) and Ballard & Montgomery (2017) in their critique of the robustness of Umbrella reviews were encountered in this study. These challenges included primary studies overlapping between reviews and appearing in more than one review, and a mismatch between the scope of the systematic review being examined and the research question of the Umbrella review. Seven of the 12 reviews had a remit for obesity prevention rather than healthy eating as a primary outcome. Furthermore, the heterogeneity of the reviews and the assessment of insufficient information in three reviews precluded an evaluation of the quality of the research through the use of GRADE. This was compounded by the difficulty for the systematic reviews to apply GRADE or a meta-analysis for the same reason. To address these challenges, the primary studies which appeared in more than one review were identified and the extent of overlap considered. Moreover, in the data extraction stage, within these accepted reviews, only primary studies which met the scope of the Umbrella review were included, strengthening confidence in the findings. Mapping the overlapping primary studies reassured the authors that the search strategies were through and demonstrated consistency between reviewers. To further ensure methodological strength, the scope of the Umbrella review was limited to only those reviews where diet-related behaviours and measures were reported separately, PRISMA guided the search strategy and two validated tools were used to assess the quality and risk of bias of the reviews and to standardize data extraction . The conInterventions promoting healthy eating positively influence children’s dietary food patterns. Although environmental-level and individual-level determinants of healthy eating are impacted by centre-based interventions, these effects are not consistently translated to changes in children’s diet-related behaviours or anthropometrics as a measure of preventing obesity. Positive outcomes can be further strengthened with parental involvement and engagement, and multi-level, multi-component strategies are recommended. Comprehensiveness may, however, affected feasibility and fidelity negatively when enacted by end-users; therefore studies on existing interventions implemented by end-users are recommended. Meta-analysis and stronger study designs are called for but are often not feasible in the real world of childcare. Therefore the translation of research or expert-led interventions into practice warrants further qualitative exploration of implementation drivers and barriers with end-users. This understanding and end-user involvement may contribute to the sustainability of interventions which is rarely reported.The summarised findings and recommendations from this Umbrella review can inform child-health directed policies and practices. Based on the evidence, public health effort is warranted to support healthy eating interventions and practices in centre-based childcare. By incorporating multi-level and multi-component interventions into routine practices and extending this across the home and childcare setting, healthy food preferences and dietary-related behaviours can be influenced. More successful interventions require high levels of parental engagement, the use of behaviour change strategies and a focus on building the capacity of educators, children and parents.


The few studies that have examined the relationship between midlife cardiovascular disease risk and longer-term costs have differentiated risk using a small number of risk categories. In this paper, we illustrate the advantages of a continuous-valued score to examine the relationship between risk and longer-term costs: the Framingham 10-year coronary heart disease risk score.Our study cohort consisted of 1333 Second Generation Framingham Heart Study participants enrolled in fee-for-service Medicare for at least 8 quarters and who had a risk score assessment between age 40 and 50 years. We used generalized linear models to examine the relationships between quarterly Medicare costs and risk scores.Using risk categories defined by the Framingham score, the cost differences between a low and high risk group were 40% to over 200% greater than differences in comparable studies using a small number of risk categories. A continuous-valued score facilitates comparison of the cost consequences of impacting risk score changes. For example, an intervention that is able to reduce a person’s score change between midlife and later-life from the 75th percentile to the 25th percentile would result in almost a 20% reduction in longer-term costs. In contrast, an intervention that is able to reduce a person’s midlife score from the 75th percentile to the 25th percentile would result in a 38% reduction in costs.A continuous-valued risk score has advantages compared to defining risk based on a small number of risk categories. The American Heart Association reports that 92.1 million adults (>1 in 3) have cardiovascular disease (CVD)[et al. analyzed costs that occurred over an 11-year period beginning about 13 years on average after risk assessment [et al. examined costs that occurred up to 23 years after risk assessment [et al., expanding and extending analysis of the Daviglus et al. cohort, analyzed average annual costs that occurred on average beginning 14 years after risk assessment and extended over a 26-year period [Despite the importance of CVD, very few papers have examined the economic implications of CVD risk profiles –8 and nosessment ; and Allr period . UnderstThe Framingham Heart Study (FHS) has been invaluable in identifying risk factors for CVD and has contributed to a wide range of interventions to reduce risk and improve population health . Recentlet al. [et al. [et al. [Daviglus et al. , Willis  [et al.  and Alle [et al.  grouped et al. [The CMS data included Medicare enrollment information and expenditures (referred to as Medicare costs) between 2000 and 2008 for 3849 Framingham Heart Study participants: 740 from the Original Framingham cohort, 2796 from the Second Generation cohort, 61 from the Third Generation cohort, and the rest from special cohorts. Risk scores were calculated from exams done between 1971 and 2007. Midlife CHD risk score  and sufficient cost data were available only for the Second Generation participants, and so our initial sample was 2796. From this sample, we excluded 672 people who were continuously enrolled in a Medicare managed care program (since cost data were unavailable for them), and those who had not bought into Medicare Part B (since they were likely still working and had other insurance); 67 participants who were under age 65 years but eligible for Medicare because of disability; 15 participants who had only 2 quarters of cost data in the last 6 months of their life; following the approach of Daviglus We required at least 8 quarters of cost data to allow averaging of any post-Medicare eligibility spending “bump” at age 65 years . Also, for the many people in our data set whose first Medicare costs were after age 65 years (age at time of first costs was 68 years or older for 50% of the participants), we did not have prior enrollment information. These may have been people on a spouse policy or in a Medicare managed care program that changed to fee-for-service because of increased flexibility to obtain various types of treatment. The 8 quarter criterion allowed the costs to be averaged over a long enough period to reduce the impact of any initial eligibility increase in spending. The higher average quarterly costs of those without 8 quarters of data  suggestsWe used the Framingham CHD 10-year risk prediction score  as a wayIn our main analyses, we used the midlife score, which was the risk score assessed at the exam between age 40 and 50 years that was closest to age 45 years. As a sensitivity analysis, we considered a wider age range, 35–55 years, to define midlife. We also considered a “later-life” score, defined as the risk score assessed closest to but before age 65 years. From the 2 risk scores, we created “score change”, defined as “later-life risk score minus midlife risk score” in order to examine the extent to which people who were more successful in limiting the natural increase in risk score due to aging had lower costs.CMS cost data between year 2000 and 2008 included the following payments: inpatient, institutional outpatient facility (outpatient file), distinct physician office (carrier file), nursing home, home health care, and hospice. We adjusted the cost data for inflation using the Medical Care Consumer Price Index with 2000 as the base year . Also, bFor descriptive purposes, we reported percentages for binary variables, and mean, standard deviations and percentiles for continuous variables. We ran generalized linear models (GLMs) with a gamma distribution and log link, and used robust standard errors. Based on the results of the Park test, as a sensitivity analysis, we also ran models with a Poisson distribution. The dependent variable was “average quarterly cost”. To reduce the impact of outliers, we top-coded the cost data by replacing any quarterly costs more than 3 standard deviations from the mean with a value equal to 3 standard deviations above the mean. Twenty six cases were top-code to a value of $13,880. As noted above, for all models, we included age at year 2000 as an independent variable. We ran models with the following additional independent variables: Model 1: midlife CHD risk score; Model 2: midlife CHD risk score components—sex, age at midlife exam, average systolic blood pressure, average diastolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, smoking status (yes/no), and diabetes mellitus (yes/no); and Model 3: midlife risk score and “later-life risk score minus midlife risk score”. We converted the coefficient for each variable into the percentage change in expected quarterly costs per one unit increase in the variable. For continuous variables, one unit corresponds to one standard deviation (SD); for binary variables, one unit equals one. The percentage change is calculated as exp − 1. As a summary statistic of model performance, we reported the correlation between predicted and actual quarterly costs and a 95% confidence interval, which was estimated from 600 bootstrap replications.p < 0.05 in a two-tailed test. Analyses were done between 2013 and 2016. The Framingham Heart Study was reviewed by the Boston University Medical Campus Institutional Review Board (IRB) and all participants signed informed consents. The study reported here, which did not require additional informed consents, was approved by the Boston University Charles River Campus IRB, protocol number 3171E on April 29, 2013.We conducted our analyses using Stata Statistical Software: Release 13, StataCorp, College Station, Texas. A variable was considered statistically significant if For the main exclusion reasons, average midlife risk score was not statistically significantly different for those in fee-for-service compared to those in managed care or without Medicare Part B (672 participants); or for those with at least 8 quarters of cost data compared to those without 8 quarters of data (314 participants), though for the latter comparison, average quarterly costs were significantly higher for those with less data see . The midWe reported the descriptive statistics on risk scores calculated from the components, as well as costs and the time participants were followed in i.e., the percentage change in quarterly costs for a one SD change in the variable) is the same as in Model 1. For the binary variables, the coefficient is the percentage increase in quarterly costs when the value of the variable changes from 0 to 1. Only 4 of the 8 risk score component variables were statistically significant: age, HDL cholesterol level, current smoking and diabetes. The impact of diabetes, which has a coefficient of 109.8, indicates that compared to somebody without diabetes, someone with diabetes has quarterly costs that are almost 110% higher. The correlation between predicted and actual costs was slightly higher than when the risk score components were used as the independent variables, but the 95% confidence intervals for the correlations were wide and the differences were not statistically significant.Model 2 shows results when the risk factors that comprise the CHD score were included as separate variables. Interpretation of coefficients associated with the continuous variables  was associated with a 12.2% increase in average quarterly costs. Someone of average age at 2000 with an average midlife risk score and whose change score was at the 75th percentile had predicted quarterly costs of $2431, 20% more expensive than someone whose change score was at the 25th percentile (which was 6 points lower).i.e., Model 1), as shown in We estimated the average quarterly cost and average midlife risk scores for cases in different percentiles of the distribution of predicted costs from the model with age at 2000 and midlife risk score  and higher average quarterly costs ($2634 vs. $2535). Using Model 1, the increase in average quarterly costs associated with a one unit increase in midlife score (3.84 points) was 28.2% . However, almost all of the increase (1.6% of the 2.0%) is because “one unit” (the SD) is larger. Using the Poisson distribution instead of the gamma distribution had no substantive impact on results.In sensitivity analyses, when we expanded the definition of mid-life from age 40–50 to age 35–55 years, the eligible cohort increased from 1333 to 1472. The expanded cohort was somewhat less healthy, with a midlife risk score of 3.19 [et al. (2017)[et al. Over 25,000 study participants were placed into one of four risk categories (% of participants in each category are shown in parentheses): favorable levels on all risk factors (6% of the sample), one or more elevated risk factors (19%), 1 high risk factor (40%) and 2 or more high risk factors (35%). Median annual costs for those in the highest risk group were about $5500 higher than those in the lowest risk group. The difference in average quarterly costs of someone in the 10% of our sample with lowest risk compared to someone in the 40% of the sample with highest risk was $1936 [As noted, there have been two studies examining future costs occurring many years after CVD risk assessment. Daviglus l. (1998) analyzedl. (2017) expandedk factor 0% and 2 For organizations that manage population health, a continuous-valued risk score allows flexibility in identifying risk groups , have not found significant impacts, similar to findings from rigorously designed nonrandomized studies. He went on to note the need to “innovate, test and evaluate novel designs” (p 1463) and suggested the potential value of more targeted approaches that focus on those individuals with elevated risk. As illustrated above, a continuous-valued risk score like the Framingham risk score facilitates identifying these particularly high-risk groups. One increasingly used approach to encourage behavior change is to offer financial incentives. However, as implied by the title of a recent JAMA Viewpoint article by Thirumurthy et al. [The challenge is how to design programs to most effectively impact risk behaviors. In a recent randomized controlled trial of a workplace wellness program, Song and Baicker  found thy et al. —“The uncThere are limitations to our study. Though throughout the paper the discussion focuses on cardiovascular risk factors, we used the Framingham Coronary Heart Disease 10-year risk score. The reason for this is that the Framingham database available to us did not contain certain variables necessary to calculate the Framingham CVD risk score . Nonetheless, the risk factors in both scores are largely similar. If anything, it is likely a continuous-valued CVD risk score would have provided even better risk group differentiation than that found using the CHD score.The sample size in our study was relatively small and included mostly whites from a largely middle class community in New England. Also, we examined a relatively healthy sample at both midlife, as shown by the lower midlife risk score for most of the inclusion criteria, and at later-life, since the latter group excluded individuals that developed CHD between their midlife and later-life score. Although midlife scores were similar for those in fee-for-service Medicare and those not, their resource utilization patterns were not necessarily similar. The generalizability of results to those not in Medicare fee for service is thus uncertain. Finally, like all observational studies, we cannot rule out residual confounding and have not established a causal relationship between risk scores and costs.Our study confirmed the longer-term Medicare costs of unfavorable CVD risk factor levels at midlife. However, it also illustrated the advantages of a continuous-valued risk score as opposed to categorizing people into a small number of categories based on the presence of combinations of risk factors. With the Framingham score, we were able to better differentiate high and low risk groups with larger cost differences; determine the higher costs associated with increasing risk, thus providing a basis for more appropriately targeting interventions of different intensity for those in different risk groups; and, most important, associate a risk change score with longer-term costs and thus demonstrate the potential cost savings of interventions that limited the increase in risk score between midlife and later-life.supplemetal


Rodents are widespread animal models in spinal cord injury (SCI) research. They have contributed to obtaining important information. However, some treatments only tested in rodents did not prove efficient in clinical trials. This is probably a result of significant differences in the physiology, anatomy, and complexity between humans and rodents. To bridge this gap in a better way, a few research groups use pig models for SCI. Here we report the development of an apparatus to perform biomechanically reproducible SCI in large animals, including pigs. We present the iterative process of engineering, starting with a weight-drop system to ultimately produce a spring-load impactor. This device allows a graded combination of a contusion and a compression injury. We further engineered a device to entrap the spinal cord and prevent it from escaping at the moment of the impact. In addition, it provides identical resistance around the cord, thereby, optimizing the inter-animal reproducibility. We also present other tools to straighten the vertebral column and to ease the surgery. Sensors mounted on the impactor provide information to assess the inter-animal reproducibility of the impacts. Further evaluation of the injury strength using neurophysiological recordings, MRI scans, and histology shows consistency between impacts. We conclude that this apparatus provides biomechanically reproducible spinal cord injuries in pigs. SCI often results in devastating neurological deficits with a major impact on the quality of life. The worldwide incidence of traumatic SCI varies substantially between different reports: It ranges from 3.6 to 1,009 patients per one million inhabitants . MoreoveIn the last few decades, the scientific community has exerted substantial efforts to overcome the consequences of SCI. An increasing number of promising therapeutic approaches have been developed in animal models, predominately in rats or mice , 7. RodeSince there is no commercially available impactor to generate SCI in large animals, we describe the development of an apparatus to perform biomechanically reproducible SCI in pigs weighing 25–50 kg. We show the iterative steps in this development, particularly focusing on the reliability and versatility of the contusion/compression device and the different surgical aspects. The multimodal sensors, the electrophysiology recordings, and the postoperative and post-mortem analyses show that our system generates reproducible acute injuries. The development of a reliable apparatus and the implementation of a surgical protocol is an essential step toward the establishment of a completely characterized animal model.in vivo experiments to determine the reproducibility of impacts. In complement to the spring-load impactor we developed tools to ensure reproducibility of the impacts and to ease the surgery.We developed two different impacting systems. During Phase 1 of the study, we produced a weight-drop impactor inspired by the early work of Allen , and itsThe animal experiments were approved by the Norwegian Animal Research Authority . The FELASA guidelines were followed. In compliance with these regulations, all efforts were made to minimize the number of animals used and their suffering. All surgical procedures were supervised by veterinarians. The experiments were performed on 20 male or female domestic pigs that weighed between 25 and 50 kg  The pigs used for this study had a genetic background composed of 50% Norwegian land pig, 25% Norwegian Yorkshire, and 25% Duroc. In addition, seven pig cadavers were used to test the instrument prototypes.All instruments engineered for this study were produced at a local workshop facility. The sketching was performed using the 3D CAD design software .For Phase 1, the weight-drop impactor is composed of guiding pipes of different lengths , in whicFor Phase 2, the spring-load impactor  is builtThe lock/release mechanism is performed through a stopper bar that keeps the piston in place when armed  contains two large rubber bands to support the pig. They are fitted on T-shaped elements. Their position may be adjusted in both the rostro-caudal and left-right axes by a system of rails and screw-locks . If desired, they may be positioned further apart to leave the abdominal region free —this requires the use of additional lateral adjustable supports to avoid lordosis of the vertebral column . They are composed of rods terminated by a large plastic disc to disperse the pressure. To avoid skin sores, gel pads are positioned between the disc and the skin of the pig. In a later stage of engineering, we replaced these lateral supports by a suspension system (further described below). Since the table is constructed of an aluminum bracket with rails, it enables the possibility to affix multiple elements, such as an articulated arm, to support the impactor.For a better positioning of the pig, we produced an operating table inspired by previous descriptions , 27. It Figure 7); it is composed of a link element  mounted on a rod fitted onto pedicle screws. Two plates (one straight plate and one bended plate) are attached to the link and glided under the spinal cord to enclose it . Flexion of the vertebral column during the impact is prevented by the fusion of three adjacent segments with pedicle screws and titanium rods . To obtain a plain contact between the dura and the piston, we straightened the vertebral column with a suspension system . It is composed of a stand attached to the pig-operating table with screws . Suspension rods terminated by a hook can be positioned and affixed to the stand by a locking system . As detailed below, the suspension rods are hooked to surgical implants to exert a vertical pull on the vertebral column .The reproducibility of the biomechanical parameters of the impacts depends on several variables. In this study, we addressed four main factors: (1) the resistance of the surface underneath the spinal cord; (2) the capacity of the spinal cord to “escape” the impact by sliding to the side; (3) the absorption of energy by flexion of the spinal column during the impact; and (4) the angle between the piston and the spinal cord. To gain control over (1) and (2), we engineered a support system that encloses the ventral and lateral aspects of the spinal cord  diluted in 0.1 M phosphate buffered saline (PBS). The load sensor measured the impact force, while the movement of the plunger was recorded at 5,630 Hz with a NX3-S1 high-speed video camera  fitted with a macro objective . For the analyses of these recordings, we used ImageJ  and the e impact . For eacFor Phase 2, the spring-load impactor, we performed a series of impacts (10 impacts for each setting) on surgical gel pads . The tension adjustment screw was set to a specific number of turns. This number of turns is correlated with the compression distance  measuredFor Pigs 1–12, we used anatomical landmarks—e.g., the scapula and counting of the ribs to identify the thoracic level. From Pig 13 onwards, we performed preoperative X-ray  and inserted a 22 G needle in the paravertebral musculature as a landmark. Once the level was confirmed, the needle was removed and the skin labeled with a permanent marker.TCO2, SpO2, and body temperature, were monitored using a multi-parameter monitor . In addition, the anesthetic depth was continuously monitored. It should be noted that the temperature at the start of the surgery was 38 ± 0.9. In all the pigs, it rose along with the surgery by 0.98 degrees ± 0.77.All anesthetic procedures were supervised by a trained veterinary anesthetist. For premedication, the pigs received an intramuscular injection of ketamine 10 mg/kg, midazolam 1.0 mg/kg, and dexmedetomidine 15 μg/kg prior to the catheterization of the auricular vein. After preoxygenation, anesthesia was induced with propofol intravenously to effect. After endotracheal intubation, intermittent positive pressure ventilation was initiated. Anesthesia was maintained with propofol 5–10 mg/kg/h, ketamine 5 mg/kg/h, and dexmedetomidine 4–8 μg/kg/h. Three to five minutes prior to the SCI, a bolus of rocuronium 1.2 mg/kg was injected intravenously to prevent any spontaneous movement. Physiological parameters, including heart rate, ECG, invasive arterial blood pressure, EFor somatosensory evoked potentials (SSEP) recordings, we stimulated the median and tibial nerves on each side using 13-mm subdermal needle electrodes 27 G, . SSEP were acquired from SCI 5–12 and 17–19 with an ISI IOM system portable . The cranial electrodes were subdermal needles  or screwThe surgery was performed by a trained neurosurgeon. The pig was placed in a prone position on the operating table. Under deep anesthesia, a 10-cm posterior midline incision was made between T8 and T12. Mobilization of the paravertebral muscles was performed with electrocautery, while diathermy was used to control haemostasis. The spinous processes, transverse processes, and the laminae of T9-11 were exposed.For Phase 1 experiments (SCI 1-9), we removed two spinous processes T10 and 11, but the lamina remained intact. The laminae were flattened with a high-speed diamante drill  to enable the positioning of the lamina plate . We remoFor Phase 2 of our study (SCI 10–20), we developed the spring-load impactor system . This sy10A–C). Two titanium rods were attached and secured to the pedicle screws. A link element designed for this purpose . With the help of anatomical landmarks, we inserted pedicle screws in T11 and T12 on the left side and in T11 and T13 on the right side of the pig , 10A–C.  purpose  was moun purpose . Nerve r purpose . The spr purpose . One min purpose . The susin vivo MRI scans (SCI 1–7), an active drain  was positioned, haemostasis was checked, and the muscle and skin were sutured using Monosyn sutures . The pig was disconnected from the automated ventilator and connected to a manual ventilation system for its transportation to the MRI facility. Anesthesia was maintained by propofol in combination with ketamine and vital parameters were monitored. T1 and T2-weighted images were acquired with a 3 T Philips MR Imaging DD 005. We also performed two CT scans (BrightSpeed S system) to confirm the laminectomy level and the screw positioning (SCI 18 and 19).For While the pig was deeply anesthetized, the laminectomy was extended to four levels. A neuromuscular blocker (Rocuronium 1.2 mg/kg) was injected (i.v.) to prevent any uncontrolled movement. The thoracic roots were inspected and no macroscopic damage was detected. Furthermore, the roots were cut and the spinal cord was transected at the rostral and caudal borders of the laminectomy. It was subsequently immersed in ice-cold PBS and maintained at 4°C until further dissection and tissue fixation were performed. The pig was euthanized by an i.v. injection of potassium chloride.http://free-d.versailles.inra.fr). For quantifying the volume of bleeding, MRI images were converted to 8-bits images and the threshold (0–60) was applied before pixel counting was done.The dura was removed and all rootlets were cut. The spinal cord was immersion fixed in 4% (w/v) paraformaldehyde-PBS for 5 days at 4°C. Two to four days prior to high-resolution MRI scanning, the spinal cord was rinsed in PBS, incubated in 1% (v/v) gadolinium  diluted in PBS, and maintained at 4°C, as previously reported for non-human primate spinal cords . Prior tFor histology, the spinal cord was embedded in paraffin , sectioned at 5–7 μm, mounted on positively charged slides, and then, processed for labeling with 0.1% (w/v) luxol fast blue diluted in 95% ethanol and 0.5% (v/v) glacial acetic acid for 2 h at 60°C. The differentiation was performed using 0.05% (w/v) of lithium carbonate, freshly dissolved in water, followed by a step in 70% ethanol. The sections were rinsed in water, incubated in absolute ethanol and xylene and coverslipped using Eukitt (Sigma-Aldrich).U-test (significance at p < 0.05). The calculations were performed using Libreoffice Calc (https://www.libreoffice.org/) or OriginPro 9.2 .The data presented indicate mean values (μ) with error bars that represent the standard deviation (σ). In the text, the coefficient of variation (CV) given as a percentage: CV = (σ/μ) × 100 is used to assess the dispersion of the data. Statistical comparison of the mean was done with the Mann–Whitney The system was tested on 10% gelatin because its surface has similar properties as the spinal cord. Such properties include consistency and deformation. However, there are several difficulties in using gelatin. If the layer is too thin, the plunger perforates the gelatin and hits the bottom of the dish. Also, the force value recorded is too high and unreliable. Thus, it is necessary to use a thick layer. However, the thickness also determines the amount of energy absorbed. It is, therefore, crucial to use a standard thickness for all impacts. In addition, a specific area of gelatin and its surrounding area may only be used once because it is damaged by the impact.p < 0.001, p < 0.001. In line with this, the CV were low, with 6.3% for the 44-cm, 5.4% for the 34-cm, and 4.8% for the 24-cm pipes. In contrast, the values for the displacement, speed, and duration data were relatively close to each other and in some case overlapped between the different pipes. When comparing these values for the 44 and 34-cm pipes or for the 34 and 24-cm pipes, there was no significant difference (p > 0.05). However, there was a significant difference (p < 0.01) between the displacement values from the 24-cm  and 44-cm  pipes. Similar results were obtained for the impact speed for the 24-cm  and 44-cm pipes . These mean speed values were significantly different at p < 0.001. The mean impact duration for the 24-cm pipe was 10.5 ms and CV 16.8%; it was 7.9 ms and CV 5.3% for the 44-cm pipe. These mean speed values were significantly different at p < 0.05. A radar plot for each impact shows the relationship among the force, displacement, speed, and duration . For convenience, we defined this value to be 100% and expressed further volume values in p < 0.025) reduction in these volumes was identified at the epicenter of the injury with a volume of white matter at 10% (CV 126%) and a volume of gray matter at 0.05% (CV 125.7%). At the epicenter, the CV exhibited substantial variations between the different samples. However, these volumes are so small compared to the uninjured tissue that such variations are almost irrelevant.Post-mortem high-resolution 9.4 T MRI  scans (SCI 14–17) indicated a severe and broad damage area in which tissue destruction coexists with multiple intramedullary bleedings . This fi3 with a CV that represented a variation of 15.4%. The cumulative loss of the gray matter was 31.8 mm3 with CV at 28.2%.The mean cumulative tissue loss  for the Substantial intra-spinal bleeding is observed not only at the injury area, but also in the perilesional zone . Pixel aSeveral types of SCI have been experimented in pigs and may be categorized as follows: (1) compression injuries using clips –40 or coin vivo. We encountered several issues. Variations in the thickness and wideness of the lamina created problems in positioning the lamina plate. Even with different plate sizes, the variation in the distance between the lamina and the dura was a recurrent problem to place the plunger in a proper position (midline and surface of dura). Hence, our concept of the lamina plate that was intended to rigidify the vertebral column and guide the plunger was riddled with difficulties. This was worsened by the opacity of the metallic pipe which denies visual control over the plunger. It also created difficulties to record some of the impact biomechanical parameters with a high-speed camera. An attempt to use a transparent plastic pipe was not successful because of frictions, which influenced the impact reproducibility. A glass pipe might have solved this issue. However, working with glass presented other problems for our local workshop and therefore this idea was abandoned. However, the weaknesses of this system included the reproducibility of the impact (discussed below) and the implementation of a static compression, following the initial impact to mimic traumatic SCI in a better way. Lee et al.  easy handling (2) reproducible impacts, (3) adjustable impact strength, (4) the possibility to apply a static compression following the initial impact, (5) better characterization of the impact with more sensor feedback, and (6) visual control for the positioning of the impactor tip on the dura. The pipe and the weight were replaced by a piston system driven by the mechanical power of an expansion spring. This approach presented multiple advantages: It is a one-piece impactor, which is easier to handle. Moreover, it does not require any assembly during the surgical procedure. Nothing prevents the visual control of the position of the piston tip on the surface of the dura. The system is suitable for use with a high-speed camera. Although for the sake of simplicity and precision, we preferred a laser displacement sensor. In addition, the spring-driven impactor can produce multiple grades of injury strength because of the adjustment screw. This is in contrast to the weight-drop system in which the impact strength depends on the length of the pipe. This was a restriction on the choice of the injury grade. An additional advantage of the spring-load impactor is that it provides a simple solution to combine a dynamic impact with a prolonged static compression. This static compression may be adjusted to a desired force or displacement after the impact. It is also possible to perform a sole stepwise spinal cord compression injury by positioning the impactor on the dura and adjusting the screw until the desired compression force or displacement is obtained. In addition, it is possible to vary the duration of the static compression to influence the injury severity and the balance between the contusion and compression components. It is also possible to mix strong contusion with weak static compression or vice versa. This versatility is an advantage for testing the robustness of a treatment. Initially, it is natural to study a defined experimental treatment with a defined injury characteristic. However, another important step would be to test the treatment in a variety of situations to characterize its indication/limits. For example, it has been suggested that the neuroprotection provided by hypothermia is more pronounced for compression with a certain degree of ischemia than for contusion injuries –55. ThisThus, the functionality and versatility of this spring-load impactor makes it an excellent tool to generate different graded SCI in large animal models with characteristics superior to those of our original weight-drop impactor.in vivo. This is critical for SCI models, particularly for demonstrating the efficacy of a preclinical treatment. In addition, a high degree of reproducibility contributes to animal welfare because it reduces the number of animals necessary for significant findings. Furthermore, experiments performed in large animals are long, expensive, and require more human resources. It is, therefore, difficult to compensate for varying degrees of injury with a larger number of animals. In line with a recent study that shows substantial differences between in vivo and ex vivo impact characteristics  may influence the impact strength. A variable is the cushion effect of the CSF. Pigs and other larger mammals have a larger CSF space than rodents , 57. FurOverall, we showed that these standardization efforts were successful, although they globally increased the complexity of the procedure. However, for experimentations on a large animal model for spinal cord injury it is necessary to work with a trained surgeon. It can be a neurosurgeon, an orthopedic surgeon, a trauma surgeon, and a veterinarian trained in spine surgery. In these circumstances, the additional steps we presented do not present any obstacle.in vivo. It is the first step toward the establishment of a complete animal model. The characterization of the biomechanical properties of the impact provides important information about the generation of the injury. In addition, SSEP and MRI are useful tools that, together with the impact properties, present a comprehensible initial characterization of the injury. However, they only show a snapshot of the situation, and the development of the secondary injury will likely aggravate the functional outcome. Therefore, further experiments on chronic animals using different longitudinal outcome measurements, such as motor behavior, electromyogram recordings, and the study of autonomic dysfunction, will be necessary to obtain a full characterization of the model. We will also need to evaluate the stability of the column because of the surgical procedure. It is possible that we would have to leave the implants to avoid kyphosis in a chronic animal. In fact, this may present the advantage to better model the situation of traumatic SCI in humans. However, such a study is beyond the scope of this paper that only focuses on the development of tools and surgical procedures to generate injuries that are as reproducible as possible. However, this may be the most important step toward the generation of a reliable model. The injury we reported here is strong. With the development of the secondary injury process, it is possible that the severity of this trauma will be beyond any possible repair. So, further experiments will also be necessary to identify the appropriate injury strength that can be used for assessing treatment effects. This will be possible because our impactor can be adjusted to provide multiple injury strength.This study, describes the engineering of an apparatus and the establishment of surgical procedures that result in the generation of reproducible spinal cord impacts MZ and J-LB initiated the study, designed the experiments, and designed the different apparatus. MZ performed the surgical procedures. AL, VB, and HH established and performed the anesthesia procedures and the monitoring of vital parameters. EK and J-LB performed intra-operative electrophysiology. LZ and J-LB performed MRI scans. MZ and J-LB wrote the paper, prepared the figures, and revised critically manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tetraspanins are a class of evolutionarily conserved transmembrane proteins with 33 members identified in mammals that have the ability to organize specific membrane domains, named tetraspanin-enriched microdomains (TEMs). Despite the relative abundance of different tetraspanins in the CNS, few studies have explored their role at synapses. Here, we investigate the function of TSPAN5, a member of the tetraspanin superfamily for which mRNA transcripts are found at high levels in the mouse brain. We demonstrate that TSPAN5 is localized in dendritic spines of pyramidal excitatory neurons and that TSPAN5 knockdown induces a dramatic decrease in spine number because of defects in the spine maturation process. Moreover, we show that TSPAN5 interacts with the postsynaptic adhesion molecule neuroligin-1, promoting its correct surface clustering. We propose that membrane compartmentalization by tetraspanins represents an additional mechanism for regulating excitatory synapses.  •TSPAN5 is expressed in pyramidal neurons and localizes mainly to dendritic spines•TSPAN5 interacts with neuroligin-1 and promotes its clustering•TSPAN5-neuroligin-1 complex is fundamental for dendritic spine maturation Moretto et al. demonstrate that TSPAN5 controls the maturation of dendritic spines by promoting the clustering of neuroligin-1. These findings provide proof of principle that compartmentalization of transmembrane proteins through tetraspanins can represent an additional level of regulation of synapse formation and function. Tetraspanins are a class of transmembrane proteins evolutionarily conserved in metazoans. They share a common structure: four transmembrane domains (TMs), a small extracellular loop (SEL) and a large extracellular loop (LEL), and intracellular N and C termini . TetraspThe role of these proteins in membrane domain assembly and in cell-cell adhesion supports a possible function in synapse formation and activity.TSPAN5 belongs to the C8 subgroup of tetraspanins, characterized by eight cysteine residues forming disulfide bridges key for proper protein folding. Structure-function studies found TSPAN5 to act in cell fusion during osteoclastogenesis  and reguTspan5 mRNA in mice revealed highest expression in the brain  clustering without strongly affecting excitatory synapse density or functionality.Dendritic spines, small dendritic protrusions where the postsynaptic compartment of excitatory synapses is formed, are typically sorted by morphology as thin, stubby, and mushroom. Their maturation process is complex and still debated, but “mushroom” spines are considered the final mature morphological subtype .NLG1, a major adhesion molecule of excitatory synapses , has rolOur data reveal TSPAN5 as a regulator of synapse organization and support the role for membrane compartmentalization of NLG1 in this process.TSPAN5 has not been studied in neurons; thus, we confirmed its expression in mouse brain lysates obtained with strong detergents (Triton X-100 and NP40) . At later stages, TSPAN5 localizes to filopodia and immature dendritic spines  markers  or a bicistronic construct carrying Sh-TSPAN5 and human TSPAN5-GFP cDNA resistant to Sh-TSPAN5 (rescue). In Sh-TSPAN5-transduced neurons, a significant reduction of TSPAN5 expression was observed. The rescue significantly restored TSPAN5 levels, with partial cleavage splitting TSPAN5-GFP fusion protein (Tspan5 mRNA. No changes were found on other tetraspanin transcripts (TSPAN7 and CD81) , shRNA targeting  protein F. We vernd CD81) G. TSPAN5nd CD81) H.To evaluate the role of TSPAN5 in dendritic spine formation, we transfected cultured rat hippocampal neurons before synaptogenesis (DIV 5) for analysis at different maturation stages. At DIV 12 A, we detAt DIV 14, synaptogenesis is prominent ; here weWe confirmed the excitatory nature of analyzed neurons by immunostaining for the excitatory marker CamKII-α  Figure J.We further studied TSPAN5 and neuronal function by performing an immunostaining for the excitatory postsynaptic marker PSD-95 and the presynaptic marker VGluT1 on mature neurons (DIV 18) transfected before synaptogenesis with the scrambled, Sh-TSPAN5, or rescue construct A. Surpriin vitro and in vivo . Upon TSPAN5 modulation, the frequency and amplitude of mEPSCs were not significantly changed C and 3D,We also tested presynaptic function by exposing transfected neurons to high sucrose concentration (1 M) to trigger presynaptic vesicle release . No chanFurthermore, upon TSPAN5 knockdown, the density of the VGlut1 puncta was reduced, while their average size increased D, suggesBecause tetraspanins are known to form TEMs, we wondered if TSPAN5 can organize similar structures in neurons and whether TEMs are involved in dendritic spine maturation.TEMs present a characteristic lipidic composition  that detTo investigate if these domains are key for dendritic spine formation, we examined the presence in TEMs of NLG1 and the AMPAR subunit GluA2, two crucial players in dendritic spine formation and excitatory synapses function, respectively . Both prHowever, this method is not specific for TEMs, as digitonin precipitates all cholesterol-enriched domains , which can be found in dendritic spines .Thus, we performed co-immunoprecipitation experiments in RIPA lysates from adult rat hippocampi and cortices, confirming the association of TSPAN5 with NLG1 A. We didImportantly, this interaction is not mediated by indirect association between different tetraspanins in TEMs, because the experiments were performed in the presence of Triton X-100 and NP40 in the lysis buffer .To map the TSPAN5-NLG1 interaction, we analyzed the main functional domains of tetraspanins, the surface exposed LEL and the C terminus (Ct), exposed to the cytosol B.We produced GST-LEL or GST-Ct fusion proteins C for pul139LQ141 and N165IYF168 regions  together with AP-NLG1 and BirA-ER. The AP tag, on the extracellular side of NLG1, is biotinylated by the enzyme BirA-ER in the ER . We usedWe also verified the involvement of the TSPAN5 Ct in NLG1 binding. When the Ct was truncated (ΔC), there was little precipitation of TSPAN5 compared with wild-type TSPAN5 D, and noWe also analyzed the region of NLG1 interacting with TSPAN5. We transfected HEK293 cells with TSPAN5-GFP and with HA-tagged wild-type NLG1, or NLG1 mutants with either the extracellular cholinesterase-homology region replaced with that of human acetylcholinesterase   Figure A. As expThe proportion of mushroom spines did not change between scrambled- and Sh-TSPAN5-transfected neurons A, supporWe validated this with a HA-tagged NLG1 SWAP mutant B. As expTo verify the importance of TSPAN5-NLG1 interaction, we compared dendritic spines of neurons transfected with scrambled, Sh-TSPAN5 or rescue constructs, expressing either wild-type TSPAN5 (rescue) or the DLQ or NIYF mutants C. First,The TSPAN5 mutants, unable to interact with NLG1, did not rescue TSPAN5 knockdown-mediated loss of dendritic spines B. FurtheSimilar results were obtained when we examined the morphology of the remaining spines. Neurons transfected with the DLQ mutant had fewer mushroom spines compared with scrambled-transfected neurons but higher than the Sh-TSPAN5-transfected neurons with a co-occurring increase in stubby spines. The NIYF-transfected neurons instead exhibited spine morphology similar to the Sh-TSPAN5 condition C.in vivo, we analyzed dendritic spines in brain slices from adult mice  that had undergone in utero electroporation of these constructs at embryonic day (E) 13.5. We first validated the shRNA for TSPAN5 in mouse neuronal cultures  in which the four cysteines  in the juxta-membrane region were mutated to alanines  or not (non-clustered condition)  FiguresE.We verified that adding clustered neurexin1β-Fc increased the average size of surface HA-NLG1/neurexin1β-Fc puncta without affecting dendritic spine density E.We found that non-clustered Sh-TSPAN5-transfected neurons still had a reduced proportion of mushroom spines compared with non-clustered scrambled-transfected neurons E and 6F.Overall, our data indicate that TSPAN5 is a regulator of NLG1 clustering. We found this process to control dendritic spine maturation without affecting the general assembly of postsynaptic compartments. Our findings are a proof of principle that compartmentalization of transmembrane proteins through tetraspanins is an extra level of regulation of dendritic spine formation.135DD137 and N165IYF168. Together with our data, this shows that the R135DD137 and D139LQ141 sequences specifically regulate the association of TSPAN5 LEL with ADAM-10 and NLG1, respectively. In contrast, the N165IYF168 motif is involved in multiple TSPAN5 interactions. TSPAN5-ADAM-10 interaction regulates the intracellular trafficking of the protease and the cleavage of specific targets , and another is shared with ADAM-10 (NIYF) . The modTSPAN5 has a vital role in NLG1 clustering, trapping it in TEMs and regulating its surface mobility. This phenomenon is NLG1 specific, as there was no change in N-cadherin mobility upon TSPAN5 modulation. Interestingly, super-resolution analyses have shown that the average dimension of TEMs (between 90 and 190 nm)  and NLG1Moreover, the defects in surface mobility of the GluA2 subunit of AMPARs upon TSPAN5 knockdown suggest that reducing NLG1 clustering impairs recruitment of other postsynaptic components . AlternaOur data found that TSPAN5 regulates dendritic spine maturation more than formation. Moreover, TSPAN5 downregulation did not affect the total number of excitatory synapses, because of an increase in shaft synapses.in vitro and in vivo that modulation of proteins involved in dendritic spine morphology, such as EphrinB3, nArgBP2 and Neurobeachin can lead to changes in the number of shaft excitatory synapses with no dramatic effect on mEPSCs   similar NLG1 has been shown to have a specific regulatory role on NMDAR-mediated currents , but theOur work does not exclude other processes underlying dendritic spine defects upon TSPAN5 knockdown; however, TSPAN5 silencing in neurons in which NLG1 was decreased or replaced with the NLG1 SWAP mutant defective for TSPAN5 association failed to affect the proportion of mushroom spines. Interestingly, a significant effect was seen on dendritic spine density and in the proportion of thin and stubby spines, suggestive of other TSPAN5 functions  . StudyinFinally, the effects on dendritic spine maturation caused by TSPAN5 knockdown can be rescued by forcing NLG1 clustering. This finding strengthens the hypothesis that TSPAN5 promotes NLG1 clustering and that this pathway is vital for dendritic spine maturation.maria.passafaro@in.cnr.it).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Maria Passafaro  on the care and use of animals and following the guidelines of the UCL-Institute of Neurology Genetic Manipulation and Ethic Committees under license from the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986 (Amended Regulations 2012). Animal procedures were approved by the Italian Ministry of Health (Protocol Number N° 100/2016) and by the France Ministry of Agriculture (N° 742/2014) for primary cultures from rat embryos and for In utero electroporation (Iacuc 799).2. Transfected cells used for streptavidin pulldown experiments were grown for 48 h in Neurobasal (GIBCO) medium supplemented with 2% B27 (prepared as in HEK293 or HeLa (Thermo Fisher Scientific) cells were grown in DMEM (GIBCO), supplemented with 10% FBS (GIBCO), 1% L-glutamine (Invitrogen), 0.1% gentamycin (Invitrogen) incubated at 37 °C with 5% COThe 293FT cell line used to generate the lentiviruses was grown in DMEM supplemented with 10% FBS, 1% L-glutamine, 0.1% gentamycin and 0.1% G418 (Invitrogen).−1 poly-D-lysine (Sigma Aldrich) at 75,000 per well and grown in Neurobasal medium supplemented with 2% B27   into pLVpSICOR was obtained from Addgene #11579 and modified by substitution of the CMV promoter with a human ubiquitin C (UBC) promoter in restriction sites NotI/NheI. The Sh-TSPAN5 sequence was inserted at the HpaI/XhoI sites and human TSPAN5 cDNA was subcloned from TSPAN5-GFP (Dr Rubinstein) and inserted upstream of the EGFP sequence at sites NheI/AgeI. Please note that the human TSPAN5 nucleotidic sequence differs from that of rat in the site of the ShRNA thus allowing to use it as shRNA-resistant sequence.The same procedure was used to produce the DLQ, NIYF and PLM pSICOR constructs by subcloning from TSPAN5-DLQ-GFP, TSPAN5-NIYF-GFP and TSPAN5-PLM-GFP .pSICOR-mCherry was obtained from Addgene #21907 and Scrambled or ShRNA sequences were inserted in the HpaI/XhoI sites and the cDNA of human wild-type TSPAN5, DLQ and NIYF mutants were inserted in NheI/AgeI sites.TSPAN5-RFP was produced by PCR and inserted in tDimer-RFP vector in the EcoRI site.pGEX-4T-1 constructs were produced by PCR from TSPAN5-GFP, TSPAN5-RDD-GFP, TSPAN5-DID-GFP, TSPAN5-DLQ-GFP and TSPAN5-NIYF-GFP (Dr Rubinstein) and by insertion at the BamHI/EcoRI sites.2 in H20 (200 μl per well). One volume of HEBS buffer  was added to the DNA and thoroughly mixed to produce air bubbles. The mix was added to the cells and left for 5 h before washing and changing the medium.HEK293 cells were transfected with the calcium phosphate method. Briefly, DNA (2 μg x 6-well) was mixed with 130mM CaClDIV5 rat or mouse hippocampal neurons were transfected with Lipofectamine 2000 (Invitrogen) or infected with lentiviral particles produced as previously described .2 (Sigma Aldrich) and 1 mM MgCl2 (Sigma Aldrich) at 37°C. Neurons were then exposed to PBS supplemented with 0.1mM CaCl2 and 1 mM MgCl2 with and without BS3 crosslinker  at 4°C for 10 min. Neurons were then rapidly washed first with TBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 plus 50 mM glycine (Sigma Aldrich) at 4°C and then with TBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 at 4°C prior to lysis with BS3 buffer . 3X Laemmli sample buffer was then added and the samples analyzed by SDS-PAGE and western blotting.The experiments were carried out according to Hippocampi and cortices were collected from adult Wistar rats and homogenized with glass-teflon potter in homogenization buffer . The total homogenate was centrifuged at 1,000 g for 10 min at 4°C. The pellet P1 corresponds to the nuclear fraction. The supernatant S1 was centrifuged at 10,000 g for 15 min at 4°C. The resulting pellet (P2) corresponds to crude synaptosomal fraction while the supernatant (S2) contains cytosolic components and light membranes. The P2 fraction was resuspended in homogenization buffer and centrifuged again at 10,000 g for 15 min at 4°C to wash the synaptosomes. Crude synaptosomes were loaded on top of a discontinuous sucrose gradient  and centrifuged at 150,000 g at 4°C for 2h. Purified synaptosomes were collected between 1 and 1.2 M sucrose layers. The fractions were all resuspended in homogenization buffer. 3X sample buffer was then added and the samples analyzed by SDS-PAGE and western blot by loading the same amount of proteins.For immunoprecipitation experiments, hippocampi and cortices were dissected from adult rat brain, pooled together and lysed in RIPA buffer  in teflon-glass potter, rotated for 1h at 4°C and centrifuged at 10,000 g for 30 min at 4 °C. Supernatants were incubated with antibodies at 4 °C overnight. Protein A-agarose beads  were incubated with the supernatants at 4 °C for 2 h. Beads were washed three times with RIPA buffer, resuspended in 3X sample buffer and analyzed by SDS–PAGE followed by western blotting.For streptavidin-precipitation experiments, transfected HEK293 cells were lysed in RIPA buffer , rotated for 1h at 4°C and centrifuged at 10,000 g for 30 min at 4 °C. Supernatants were incubated with streptavidin immobilized on agarose beads (Thermo Scientific) for 3 h at 4°C. The beads were first precipitated by centrifugation at 4,000 RPM for 5 min at 4°C, washed three times with RIPA buffer and then resuspended in 3X sample buffer and analyzed by SDS–PAGE followed by western blotting.For GFP-Trap precipitation, transfected HEK293 cells were lysed in RIPA buffer , rotated for 1 h at 4°C and centrifuged at 10,000 g for 30 min at 4 °C. The supernatants were first incubated with GFP-Trap magnetic beads (ChromoTek) for 1 h at 4°C and then precipitated by magnetic separation and washed three times with RIPA buffer, resuspended in 3X sample buffer and analyzed by SDS–PAGE followed by western blotting.E. coli and inducing recombinant protein expression by adding IPTG  for 2 h. Bacteria were pelleted, resuspended in lysis buffer  and rotated for 1 h at 4°C. Lysed bacteria were inserted into a dialysis membrane (cutoff 13kDa) and dialyzed for 1 h at 4°C in dialysis buffer I . The membrane was then moved to dialysis buffer II  overnight at 4°C. Membranes were dialyzed with new dialysis buffer II for 3 h at 4°C. Lysates were then collected and centrifuged at 10,000 g for 30 min at 4°C. The supernatant was incubated with glutathione Sepharose beads (Thermo Scientific) overnight at 4°C and then washed with dialysis buffer II.GST-fusion proteins were prepared by growing transformed BL21 Hippocampi and cortices dissected from adult rat brains were pooled together, lysed in RIPA buffer by homogenization in a teflon-glass potter, rotated for 1 h at 4°C and then centrifuged at 10,000 g for 30 min at 4 °C. Supernatants were incubated with glutathione Sepharose beads for 3 h at 4°C and then washed and resuspended in 3X sample buffer and analyzed by SDS-PAGE followed by western blotting.Proteins were transferred from the acrylamide gel onto the nitrocellulose  or PVDF membranes. Membranes were incubated with the primary antibodies  at room temperature for 2–3 h or overnight at 4°C in TBS Tween-20 (0.1%), milk (5%). After washing, the blots were incubated at room temperature for 1 h with horseradish peroxidase-conjugated α-rabbit, α-mouse or α-rat antibodies  in TBS Tween-20 (0.1%), milk (5%). Immunoreactive bands on blots were visualized by enhanced chemiluminescence .Mouse hippocampal cultures were prepared as reported previously . To moduFor each condition, 1.5 ug of extracted mRNA was used to synthetize cDNA using SuperScript VILO cDNA Synthesis Kit (Thermo Fisher).The target sequences of TSPAN5, TSPAN7, CD81 and α-actin (endogenous control) were amplified from 60 ng of cDNA in the presence of SYBR Green PCR Master Mix (Applied Biosystems) using Applied Biosystems 7000 Real-Time thermocycler. Primer sequences were as follows: mouse TSPAN5 Fw (AACAACATCAGAGCCTACAGAG), mouse TSPAN5 Rev (GGTTCCAATCATCAGCTCCA), TSPAN7 Fw (ACCAGTTTTATGGAGACTAACATGG), TSPAN7 Rv (AGCAGCATGCCAATCAACT), CD81 Fw (TGATGATGTTTGTAGGCTTCCT), CD81 Rv (CTCACAGGCAAACAGGATCA), α-actin Fw (AGATGACCCAGATCATGTTTGAGA), α-actin Rev (CCTCGTAGATGGGCACAGTGT)Each sample was run in triplicate, and the results were calculated using the ΔΔCT method to allow the normalization of each sample to the internal standard and comparison with the calibrator of each experiment.ARs blocker bicuculline (20 μM) to isolate mIPSCs and mEPSCs respectively. The composition of the intracellular solution was 126 mM K-gluconate, 4 mM NaCl, 1 mM EGTA, 1 mM MgSO4, 0.5 mM CaCl2, 3 mM ATP , 0.1 mM GTP , 10 mM glucose, 10 mM HEPES-KOH . Mixed AMPA/NMDA-mEPSCs were recorded in absence of Mg2+ to reduce Mg2+ block of NMDARs. Pure AMPAR mediated currents were isolated by perfusing neurons for at least 6 minutes with an NMDAR blocker (100 μM APV) to estimate the time when the AMPAR component of mixed AMPA/NMDA-mEPSCs decays. This allowed the measure of the NMDAR component of mixed AMPA/NMDA-mEPSCs. Specifically, the AMPAR component of mixed events were measured at the peak of the current while the NMDAR component were measured in a window between 10-20 ms after the AMPAR peak (adapted from N-ethyl bromide (QX-314 5 mM).Miniature excitatory or inhibitory post-synaptic currents (mIPSCs/mEPSCs) were recorded in the presence of the voltage-dependent sodium channels blocker (500 μM lidocaine). Additional blockers were added including the broad-spectrum glutamatergic blocker kynurenic acid (3 mM) and the GABAThe readily releasable pool (RRP) size was evaluated by perfusing neurons for 5 s with KRH supplemented with 1 M sucrose as previously described .Recordings were performed with a Multiclamp 700B amplifier . Pipette resistance was 2–3 MΩ and series resistance always below 20 MΩ. mEPSCs and RRP size were recorded at a holding potential of −70 mV, filtered at 2 kHz, and digitized at 20 kHz using Clampex 10.1 software . Analysis was performed offline with Clampfit 10.1 software  using a threshold crossing principle. Cells with noisy or unstable baselines were discarded.2 and 1 mM MgCl2 and fixed in paraformaldehyde /sucrose  for 10 min at room temperature or in methanol (Sigma Aldrich) for 10 min at 4°C and incubated with primary antibodies  in GDB1X solution  for 2 h at room temperature.Cultured hippocampal neurons were washed in PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 and fixed in paraformaldehyde / sucrose  for 10 min at room temperature and then blocked for 1 h with blocking solution  followed by 3 h incubation with α-pan neurexin-1 (1:250) in primary antibody solution .For neurexin staining neurons were washed in PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 and paraformaldehyde fixation.For surface staining, antibodies  were applied to neurons for 10 min at room temperature followed by a washing step in PBS supplemented with 0.1 mM CaCl42-, pH 7.4) the coverslips were incubated with secondary antibodies  in GDB1X solution for 1h at room temperature.After three washes with high salt buffer .Fluorescence images were acquired with an LSM510 Meta confocal microscope  and a 63X oil-immersion objective  with sequential acquisition setting, at 1,024 X 1,024 pixels resolution. Images were Z series projections of approximately 6–10 images, each averaged four times and taken at depth intervals of 0.75 μm.Dendritic spines were counted on all GFP positive neuronal dendritic arbor excluding the soma and classified with NeuronStudio software (NeuronStudio©) according to the following parameters: General parameters for spine identification: Length > 0.2 μm and < 3.0 μm, Max Width 3.0 μm, Stubby spines size > 10 voxels, Non Stubby spines size > 5 voxels. For spine types classifications the following logical tests were used: if Neck Ratio (head/neck diameter) > 1.100 then a spine was classified as Thin  or Mushroom . A spine is classified as Stubby if it fails at any of the precedent logical tests.Sholl analysis was performed with the NeuronStudio software using concentric circles with 3 μm radius.Puncta or clusters were calculated with Fiji software thresholding images at intensity equal to 3 X StDev of the signals.For For NLG1 clustering was induced by application of clustered neurexin1β-Fc. Purified neurexin1β -Fc  were preNon-clustered or clustered neurexin1β -Fc were applied to DIV12 neurons in media and left for two days.2 and 1 mM MgCl2 to surface stain both HA-tagged-NLG1 and neurexin1β-Fc.α-HA and α-human Fc-Alexa647 were applied for 10 min at room temperature in PBS supplemented with 0.1 mM CaClNeurons were then fixed with paraformaldehyde (4%)/sucrose (4%) for 10 min at room temperature, washed and stained with a secondary-Alexa568 antibody to detect α-HA signal.After imaging as above, both the Alexa568 and Alexa647 signals were thresholded for intensity equal to three times the StDev of the signals. Only puncta positive for both channels were considered in the analysis.All procedures were approved by the Italian Ministry of Health and the San Raffaele Scientific Institute Animal Care and Use Committee in accordance with the relevant guidelines and regulations. Electroporation in utero was employed to deliver the expression vectors to the ventricular RGCs of CD1 mouse embryos as previously described . BrieflyAnimals with GFP and tdTomato positive signals in the brain were aged to P30 and then anesthetized with 10 mg/ml Avertin and intracardially perfused with 4% paraformaldehyde/ 4% sucrose. Brains were collected, 150 μm thick slices were cut with a vibratome (Leica) and mounted on polylysine-covered coverglass (VWR) with FluoroMount (Sigma Aldrich).Secondary dendrites of around 60μm from GFP and tdTomato positive pyramidal cortical neurons were imaged with a Z-projection of 0.35 μm steps with the confocal ZEISS LSM 800 microscope mounting a 63X oil immersion objective at 1024X1024 pixels resolution. Dendritic spines were counted manually from secondary branches of the apical dendrite of layer III pyramidal neurons in the lateral somatosensory cortex.Transfected rat hippocampal cultured neurons were imaged either with monomeric streptavidin coupled to Atto-594  to imageBriefly, coverslips were mounted in an open Inox chamber (Life imaging services) and the growth medium was replaced by Tyrode solution  containing 1% globulin-free BSA (Sigma). A Nikon Ti-E Eclipse inverted microscope equipped with an EMCCD camera  and an apochromatic (APO) total internal reflection fluorescence (TIRF) 100X/1.49 numerical aperture (NA) oil objective was used for imaging. GFP positive neurons were selected and low concentrations of Atto 594-conjugated mSA (1 nM) or Atto 647- conjugated α-GluA2 antibody were added to isolate single molecules. A four-color laser bench  was connected through an optical fiber to the TIRF illumination arm of the microscope. Laser powers were controlled through acousto-optical tunable filters driven by the Metamorph software . Atto 594 and Atto 647 were excited with the 561- and 642-nm laser lines through a four-band beam splitter . Samples were imaged by oblique laser illumination, allowing the excitation of individual Atto-conjugated ligands bound to the cell surface, without illuminating ligands in solution. Atto 594 and 647 fluorescence was collected using FF01-617/73 and FF01-676/29 nm emission filters (SemRock), respectively, placed on a filter wheel (Suter). Stacks of 4,000 consecutive frames were obtained from each cell, with an integration time of 20 ms. Acquisitions were steered using the Metamorph software (Molecular Devices) in a streaming mode at 50 Hz.Images were analyzed with a custom Metamorph macro based on wavelet segmentation for localization and simulated annealing algorithms for tracking, previously described . Super-rmEOS-tagged N-Cadherin was generated as previously described . HippocaPrimary cultured neurons were surface-labeled with 100 nM of mStrav-Alexa647 in Tyrode solution for 10 min, rinsed, and fixed with pure methanol 8 min at −20°C. Neurons were permeabilized using 0.2% Triton X-100 (Sigma) for 5 min, rinsed, blocked using 0.1% BSA (Sigma) in PBS for one hour, then immunolabeled for TSPAN5 (1:50), and incubated with Alexa 532 conjugated anti-rabbit secondary antibody (Jackson Immunoresearch). dSTORM imaging of cultured neurons was performed using an inverted motorized microscope  equipped with a x100 1.49NA PL-APO objective and a perfect focus system, allowing long acquisition in oblique illumination mode, 1W 532 and 647 laser lines. Both the ensemble and single-molecule fluorescence were collected by using a quad-band dichroic filter . The fluorescence was collected using a sensitive EMCCD . 100-nm fluorescent beads  adhered to the surface of the coverslips were used as registration markers. Single-molecule localization and reconstruction was performed with automatic feedback control of the lasers using WaveTracer module, enabling optimal single-molecule density during the acquisition . The acqBiorender.com).The graphical abstract was prepared using the BioRender software (All statistical analyses were done with GraphPad Prism 6 software.Two-tailed unpaired t test was performed to assess statistical significance between two independent groups A. One-waStatistical details of the experiments can be found in the Western blots were repeated at least three times from three independent experiments. Imaging experiments on cultured neurons were done on at least three independent cultures.This study did not generate any datasets or code.


The impact of chronic cadmium exposure and slow accumulation on the occurrence and development of diabetes is controversial for human populations. Islets of Langerhans play a prominent role in the etiology of the disease, including by their ability to secrete insulin. Conversion of glucose increase into insulin secretion involves mitochondria. A rat model of pancreatic β-cells was exposed to largely sub-lethal levels of cadmium cations applied for the longest possible time. Cadmium entered cells at concentrations far below those inducing cell death and accumulated by factors reaching several hundred folds the basal level. The mitochondria reorganized in response to the challenge by favoring fission as measured by increased circularity at cadmium levels already ten-fold below the median lethal dose. However, the energy charge and respiratory flux devoted to adenosine triphosphate synthesis were only affected at the onset of cellular death. The present data indicate that mitochondria participate in the adaptation of β-cells to even a moderate cadmium burden without losing functionality, but their impairment in the long run may contribute to cellular dysfunction, when viability and β-cells mass are affected as observed in diabetes. At the end of the treatment, cells were washed with PBS and brought in suspension by incubation with 0.25% trypsin/EDTA in PBS without calcium and magnesium. Cells were pelleted at 150× g for 5 min, the pellet was rinsed with PBS, and suspended at 106 cells/mL in 50 mM HEPES, 0.7 M NaCl, 12.5 mM CaCl2, pH 7.4. The suspension was labeled with Fluoprobe 488-annexin V (Interchim) then 1 μg/mL propidium iodide (PI) for 15 min at room temperature in the dark. The stained cells were detected by flow cytometry with a LSR Fortessa™ cell analyzer  using the 488 nm sapphire laser and 532 nm compass laser for Fluoprobe 488 and PI, respectively. The corresponding fluorescence emission was measured with a 525/50 nm and 585/15 nm band-pass filters, respectively. Live cells are not labeled in this assay, whereas preapoptotic ones bind annexin V, necrotic ones accumulate PI, and doubly labeled cells are the dead ones. As an alternative method to the above labeling of cells, viability was also measured with the Cell Titer 96® AQueous One Solution Cell Proliferation Assay  in 96 well plates until adherence, then cadmium was added at different concentrations as explained above. The number of cells able to reduce the MTS tetrazolium compound was determined by recording the absorbance at 490 nm with a multi-well plate reader .To assess viability, INS-1 cells were exposed to CdCl2 for 96 h as described above. In wells in which mitochondria were labeled without nuclear staining, the cell-permeable fluorescent probe MitoTracker Red CMXRos  was added at 200 nM for 30 min at 37 °C. Cells were fixed in fresh 4% paraformaldehyde for 10 min at ambient temperature, washed twice with PBS, then cells were permeabilized using 0.2% Triton X-100 in PBS for 15 min, rinsed thrice, and blocked with PBS-Tween (1 mg/mL) BSA 5% (PBS-T BSA) for 1 h at 37 °C. Mitochondria were alternatively labeled with the primary antibody  raised in rabbit against mitochondrial aconitase (the product of the ACO2 gene) as an alternative to MitoTracker staining. The mAb was diluted 200 fold in PBS-T BSA and cells were incubated overnight at 4 °C. The cells were then rinsed thrice with PBS, and the primary antibody was reacted for 4 h at room temperature in the dark with the labeled secondary one  diluted 200 fold in PBS-T BSA. Before the end of the latter incubation, nuclear staining was performed with PI (1 mg/mL) for 20 min at 4 °C. Culture chambers were removed, and slides were mounted and sealed before microscopic observation.In immunofluorescence (IF) experiments, INS-1 cells were inoculated at 5000 cells/well on culture slides with detachable culture chambers  until adherence. They were treated with different concentrations of CdClA Leica TCS SP8 inverted laser scanning confocal microscope  equipped with a 40× Oil immersion objective was used to collect images. Laser excitation was 488 nm for Hylite Fluor 488, 552 nm for MitoTracker Red CMXRos and PI, with fluorescence emission at 500–550 nm, 575–630 nm, and 605–685 nm, respectively. The Mitotracker probe was used to cross check the images recorded by labeling aconitase: both sets of images qualitatively agreed and, since the latter were of better quality than the former, only wells in which aconitase was detected were analyzed in details. Several fields were recorded for each slide and quantitative analysis with the Image J (imagej.nih.gov) and Volocity  computer programs was carried out on all of them as follows. In a first step, tophat filtering was applied to the images recorded with the mitochondrial channel (aconitase fluorescence) in Image J to remove noise and to obtain a precise definition of the mitochondrial morphology. The filtered images were then analyzed with the Volocity software which provides morphological parameters like perimeter, area, skeletal length and diameter for each identified object. Each analyzed Cd-treatment group corresponded to tens of cells, and hundreds or thousands of mitochondrial objects. From these data, the circularity shape factor was calculated, as Equation (1).w:v) HClO4-EDTA 6.25 mM. The recovered mixture was strongly mixed and centrifuged at 12,000× g for 5 min at 4 °C. The supernatant was neutralized at pH 7 with MOPS-KOH buffer 0.3 M and centrifuged 2 min at 12,000× g. Aliquots (75 μL) of the supernatant were then mixed with 15 μL of HCl 1 M and 35 μL of 28 mM pyrophosphate buffer pH 5.75. Thirty µl of the mixture were analyzed on a Polaris 5 C18-A, Agilent S (250 × 4.6 mm) column equilibrated in pyrophosphate buffer pH 5.75 at 1 mL/min. The retention times of ATP, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) are approximately 6, 7 and 12 min, respectively, as determined by UV absorption at 254 nm.To measure the cellular content in adenosine nucleotides, cells were rinsed with PBS after the cadmium treatment as described above. Cellular perchlorate extracts were prepared with 750 μL of cold 2.5%  with 2.8 or 16.7 mM of glucose at ca. 4 × 106 cells/mL. These glucose concentrations were chosen at the lower and higher ends of the sigmoid response of β-cells to glucose. Cellular respiration was measured and, when required, 1 μg/mL oligomycin was added. The fraction of O2 consumption used for producing ATP was calculated as Equation (2).The oxygen consumption rates of INS-1 cells were measured with a temperature-controlled Hansatech oxygraph equipped with a Clark electrode and monitored with the 6 cells). Dihydroethidium (DHE) at the final concentration of 5 μM was added to the cell suspension which was then incubated 30 min in the dark at 37 °C, and cells were analyzed for fluorescence with a LSR Fortessa™ cell analyzer .To estimate the level of oxidative species present inside cells at the end of the exposure period to cadmium, INS-1 cells were grown and treated with CdCl2 as above  for 96 hTo measure the concentration of cadmium inside cells, the latter were harvested, rinsed and dry pellets were kept at −80 °C before measurements. The latter were carried out by Inductively-coupled plasma-mass spectrometry (ICP-MS) as described in details elsewhere . An aliqp < 0.05 status were recorded.The implemented statistical tests were adjusted to the design of the different experiments, the nature of the measured parameters, and the kind of comparison to be made. The Kruskal–Wallis test or One Way Analysis of Variance, depending on the data distributions, were applied to the parameters tested to vary with the cadmium exposure group. For microscopic image analysis, the Dunn’s test was used to compare each group to the one which was not exposed to cadmium. The difference of rank means, the Q test statistic, and the Since the aim of the present study is to probe the sensitivity of the INS-1 cell line upon long-term exposure to sub-lethal concentrations of cadmium, these β-cells were kept for the maximal amount of time in culture in the presence of cadmium before analysis. It was observed that a given batch of cells could not sustain growth for more than ca. 4 days before reaching high density for those not exposed or dying for the most exposed ones, i.e., without applying the stress associated with passage in suspension. Hence, 4 days was the time limit set for the following experiments. The analysis of viability after 72 h indicated that the proportion of viable cells was not significantly altered below 2.5 μM . By furtFrom the above, it was decided that only cadmium concentrations well below the median lethal dose would be worth considering in an effort to mimic chronic low-level exposure. Therefore, further data were obtained by exposing cells to concentrations up to 2 or 2.5 μM, for 96 and 72 h, respectively, these upper points being taken as limits corresponding to the onset of cell death, of less than 15% in each case.Despite the sub-lethal cadmium concentrations used in the experimental setup defined in The visualization of mitochondria by immunofluorescence staining showed that the aspect of the mitochondrial network changed after exposure of cells to doses of cadmium for 96 h . The conThe microscopic images were treated as described in the Material and Methods section. Two parameters, circularity and skeletal length, were selected for analysis, and the results of comparisons between cadmium treatment groups and control INS-1 cells are shown in One of the main functions of mitochondria is to convert energy in the form of ATP. It was thus of interest to examine whether the reorganization of the mitochondrial network observed in 2. Islets of Langerhans produce insulin in response to variations of glucose concentrations, and mitochondria are responsible for increased ATP production under these conditions. The latter was thus measured in response to increased glucose concentrations (The ATP/(ADP + AMP) ratio under standard growth conditions a was foutrations b. High  ratio as a function of cadmium exposure, is statistically significant only at the onset of cell death, i.e., at a 3 day-dose of 2.5 μM a. This rt change a. This st change a is note2 consumption devoted to ATP production which first increases as a function of cadmium exposure to reach significance at 1 μM, and then partly collapses at 2.5 μM  that eventually could become significant at higher cadmium concentrations. In this respect, mitochondrial dysfunction cannot be considered as an early sign of cadmium poisoning of β-cells, and any biomarker related to mitochondrial integrity is unlikely to provide a reliable and sensitive probe of mild exposure to cadmium.Changes of mitochondrial morphology and function in β-cells have already been correlated with apoptosis ,29. But These data are another illustration of one of the ways cells try to adapt to an environmental stress . It is lBut long-life provision of cadmium to pancreatic β-cells is likely to eventually lead to dysfunction, including of their mitochondria. The data reported herein apply to a simple cell model that has been exposed for a relatively short time as compared to the conditions that apply to long-lived animals, humans in particular. This is an obvious and important limitation of this work. However, the data suggest that non-invasive detection of cadmium in islets of Langerhans may provide a useful marker for subjects at risk of developing diabetes. Methods to detect cadmium in vivo are developing , and the


Reduced gene diversity, particularly in genes involved in immunity, may have important functional consequences. In fact, it has been suggested that reduced diversity in immune genes may have contributed to Neandertal extinction. We therefore explored gene diversity in different human groups, and at different time points on the Neandertal lineage, with a particular focus on the diversity of genes involved in innate immunity and genes of the Major Histocompatibility Complex (MHC).Genome-wide analyses of two Neandertals and a Denisovan have shown that these archaic humans had lower genetic heterozygosity than present-day people. A similar reduction in genetic diversity of protein-coding genes (We find that the two Neandertals and a Denisovan have similar gene diversity, all significantly lower than any present-day human. This is true across gene categories, with no gene set showing an excess decrease in diversity compared with the genome-wide average. Innate immune-related genes show a similar reduction in diversity to other genes, both in present-day and archaic humans. There is also no observable decrease in gene diversity over time in Neandertals, suggesting that there may have been no ongoing reduction in gene diversity in later Neandertals, although this needs confirmation with a larger sample size. In both archaic and present-day humans, genes with the highest levels of diversity are enriched for MHC-related functions. In fact, in archaic humans the MHC genes show evidence of having retained more diversity than genes involved only in the innate immune system. Ne)  .−5, similar to that previously reported for the ∼120,000-year-old Altai Neandertal (−5)  . This lotal (−5) . GeneticIt has been suggested that lack of functional variation in immune-related genes—especially in genes related to the innate immune system which is known to serve as a first defense mechanism against pathogen detection—, some of which are targets of long-term balancing selection , could hHere, we leverage existing high-quality whole-genome data from three archaic humans to test for evidence of a specific reduction of gene diversity in archaic humans that would be expected under the DPRH. We focus on comparing genetic diversity between archaic and present-day humans, and over time in the Neandertal lineage in a comprehensive set of 1,548 innate immunity genes defined by N = 17,505). We extracted the sequences that pass these filters for each individual from the whole genome VCF files and excluded genes with <2,000 callable sites from the analysis . Data processing was done using Tabix . We updated this gene list with recent InnateDB entries following our filtering scheme. This resulted in a set of 1,548 innate immunity genes. We additionally investigated the following subsets of this innate-immune gene list separately: Toll-like receptorsignalingpathway , innate immune response in mucosa , defense response , defense response to bacterium , defense response to Gram-negative bacterium , defense response to Gram-positive bacterium , defense response to fungus , defense response to virus , as well as the MHC genes (N = 14). We then defined a hand-curated set of autosomal protein-coding background genes without any reported immune function to use as a background set  for whibl genes ) as wellWe performed a gene ontology (GO) enrichment analysis to explore whether any particular functional groups of genes (GO categories) are overrepresented among the genes with the highest  or lowest (bottom-5% tail of the same distribution) SNP/FD ratios in the three archaics, or a set of three representative present-day humans . In this analysis, we only considered genes that pass our above-mentioned filters in the test individuals, and averaged the SNP/FD ratio over those individuals for each gene. For GO enrichment analyses, we used the R package “GOfuncR” . In the We estimated gene diversity per individual by calculating SNP/FD ratios in five present-day individuals from African populations , and nine present-day individuals from non-African populations . In agreement with previous observations, we consistently found significantly higher diversity in African individuals than in individuals from non-African populations . All thrNext, we tested whether genes of the innate immune system showed a similar reduction in Neandertals (when compared with present-day humans) as non-immune genes. To further investigate a putative specific reduction in innate immune gene diversity, we investigated normalized immune gene diversity by dividing mean SNP/FD ratios in immune genes by mean SNPD/FD ratios in a set of non-immune-related background genes . This further suggests that immune gene diversity did not decrease over time. We note that differences in the branch lengths leading to each of the archaic humans, which reflect the differences in ages of the specimens , do not have a substantial effect on our analyses as we do not observe a strong positive correlation between the age of archaic individuals and the normalized gene diversity .2-values of lower 95% confidence intervals > 0). Interestingly, MHC diversity is ∼47-fold higher than the background genes in archaic humans (95% CI: 32–76-fold) but only ∼7-fold higher than background genes in present-day humans (95% CI: 5–9-fold). This higher diversity in the MHC observed in archaics compared with present-day humans is driven largely by the MHC class II genes . B2M is part of the class I MHC (light-chain) but—unlike the other MHC genes—is located on chromosome 15. It is known to be nonpolymorphic in humans  show a highly significant enrichment only of GO categories related to the MHC in both the archaic and present-day humans after correcting for multiple testing  It could be caused by the old TMRCA in the MHC region  and the arger Ne . A possion rates . This ison rates . TheoretFuture sequencing of additional high-coverage archaic genomes that sample the geographic and temporal distribution of Neandertals will allow questions about the effects of gene diversity on Neandertal fitness to be addressed in greater detail.Genome Biology and Evolution online.Supplementary DataClick here for additional data file.